非洲猪瘟病毒P30-54融合蛋白在杆状病毒表达系统中的表达及反应原性研究  

Expression and Reactiongenicity of P30-54 Fusion Protein of African Swine Fever Virus in Rotavirus Expression System

作　　者：李杰[1] 张星星[2] 郭晶 张国武 王晓婷 李妍 才学鹏[3] 孟庆玲[1] 乔军[1] LI Jie;ZHANG Xing-xing;GUO Jing;ZHANG Guo-wu;WANG Xiao-ting;LI Yan;CAI Xue-peng;MENG Qing-ling;QIAO Jun(Department of Animal Science & Technology,Shihezi University,Xinjiang Shihezi 832003,China;State Key Laboratory for Sheep Genetic Improvement and Healthy Production,Xinjiang Academy of Agricultural and Reclamation Science,Xinjiang Shihezi 832003,China;Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Gansu Lanzhou 730046,China)

机构地区：[1]石河子大学动物科技学院,新疆石河子832003 [2]新疆农垦科学院畜牧兽医研究所,新疆石河子832000 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046

出　　处：《西南农业学报》2019年第3期684-688,共5页Southwest China Journal of Agricultural Sciences

基　　金：国家重点研发计划项目子课题(2017YFD0502300);兵团中青年科技创新领军人才计划(2016BC001)

摘　　要：【目的】非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种严重危害猪养殖业的急性烈性传染病。【方法】为了制备特异性强、敏感性高的ASFV血清学诊断用抗原,本研究利用Bac-to-Bac杆状病毒表达系统表达了ASFV P30-54融合蛋白基因。将P30-54融合蛋白基因克隆入杆状病毒转座载体质粒pFastBac1中,构建重组质粒pFastBac-P30-54;将重组质粒转化DH10 Bac感受态细菌进行重组,经抗性与蓝白斑筛选得到杆状病毒重组质粒Bacmid-P30-54;将reBacmid-P30-54转染sf9细胞,获得重组杆状病毒reAcMNPV-P30-54。将获得的reAcMNPV-P30-54接种sf9细胞,待细胞出现CPE时收集细胞,用间接免疫荧光试验(IFA)和SDS-PAGE进行分析重组蛋白的表达情况;通过Western blot检测重组蛋白的反应原性。【结果】在重组杆状病毒中成功表达P30-54蛋白,该重组蛋白可与ASFV多克隆抗体发生免疫学反应。【结论】证实该重组蛋白具有良好的反应原性。【Objective】African pig fever(ASF) is an acute infectious disease caused by African pig fever virus(ASFV).【Method】In order to prepare high-reactive ASFV serum diagnostic antigen, ASFV P30-54 fusion protein gene was expressed using Bac-to-Bac baculovirus expression system in this study. P30-54 fusion protein gene was cloned into plasmid pFastBac-P30-54 of rotavirus transposable vector;Then recombinant plasmid was transformed into DH 10 Bac bacteria to generate recombinant plasmid Bacmid-P30-54 by resistance and blue spot screening;The recombinant plasmid Bacmid-P30-54 was transfected into sf9 cells to generate the recombinant baculovirus reAcMNPV-P30-54. When reAcMNPV-P30-54 was inoculated with sf9 cells, the pathological cells were collected when CPE appeared and was analyzed by indirect immunofluorescence assay(IFA) and SDS-PAGE;Reaction genecity of recombinant proteins was detected by Western blot.【Result】The ASFV P30-54 fusion protein was successfully expressed by reAcMNPV-P30-54, which could specifically react with ASFV positive serum.【Conclusion】It was concluded that the recombinant protein displayed better reactiongenicity.

关 键 词：非洲猪瘟病毒 P30-54融合蛋白 杆状病毒表达系统 反应原性 

分 类 号：S858.28[农业科学—临床兽医学]